1. Technical Field
The present invention relates to a simple and correct method for analyzing lipoxygenase activity. More specifically, it relates to a simple method for analyzing lipoxygenase activity by measuring the absorbance of material produced by oxidation of polyunsaturated lysophosphatidylcholine by lipoxygenase.
2. Background Art
Lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.11.12; hereinafter called as “LOX”), a nonheme iron-containing enzyme, catalyzes the addition of molecular oxygen to fatty acids containing at least one (Z,Z)-pentadiene system to give corresponding hydroperoxides (W. L. Smith, W. E. Lands, J. Biol. Chem. 247 (1972) 1038-1047; S. Yamamoto, Biochem. Biophys. Acta 1128 (1992) 117-131). Lipoxygenation occurs when 1,4-(Z,Z)-pentadiene is oxidized by LOX.
The nomenclature of lipoxygenases is based on the specificity of the enzymes with respect to their substrates. For example, 12-LOX oxygenates arachidonic acid at carbon-12. The stereochemistry of the reaction can be specified when necessary (e.g. 12R-LOX or 12S-LOX). Where more than one enzyme have the same specificity, they may be named after the tissue in which they are found. For example, there can be platelet, leukocyte and epidermal types of 12-LOX.
Four main enzyme types with positional specificities occur in animal tissues, i.e. 5-LOX, 8-LOX, 12-LOX, and 15-LOX. The first step in the reaction involving 5-LOX is the abstraction of a hydrogen atom from carbon-7 by ferric hydroxide, which involves a proton-coupled electron transfer by which the electron is transferred directly to the iron (III) to produce a substrate radical. The structure of this radical is uncertain, as are the details of the next steps in which an oxygen atom is added, and the cis-double bond in position 5 migrates to position 6 with a change to the trans-configuration leaving the hydroperoxyl moiety in position 5. The resulting product is 5S-hydroperoxy-6t,8c,11c,14c-eicosatetraenoic acid (5-HPETE). 8-, 12- and 15-LOX operate in the same way to give analogous products. 15-LOX has a broader specificity and is able to oxidize linoleate to 13-hydroperoxyoctadecadienoate (and in part to the 9-isomer). It is also able to utilize arachidonate bound to phospholipids as a substrate hence the interest in the role of the enzyme in membrane disruption and in disease states is increased.
Lipoxygenase uses unsaturated fatty acids liberated from biomembranes by phospholipase A2 (Cirino, Biochem. Pharmacol. 55 (1998), p. 105; Farooqui et al, J. Neurochem. 69 (1997), pp. 889-901; Dennis, J. Biol. Chem. 269 (1994), p 13057). Although some of LOXs directly can oxidize certain phospholipids or triglycerides, free polyunsaturated fatty acids are preferable substrate (W. L. Smith, W. E. Lands, J. Biol. Chem. 247 (1972) 1038-1047; S. Yamamoto, Biochem. Biophys. Acta 1128 (1992) 117-131).
In certain tissues, particularly mammalian heart tissues, lipoxygenase oxidizes biomembranes or phospholipids (Heinrikson et al, J. Biol. Chem. 252 (197), P. 4913; Schalkwijk et al., Biochem. Biophys. Res. Commun. 174 (1991), p. 268; Petit et al., J. Neurochem. 64 (1995), p. 139) and the growth of reticulocytes are associated with lipoxygenase, suggesting that lipoxygenase may intervene biological reaction by influencing the variation of cell membrane structure (Brash et al., Biochemistry, 1987, 26; 5465-5471; Murray et al., Arch Biochem Biophs. 1988; 265; 514-523). A recent study reported that lipoxygenase is related with the oxidation of low density lipoprotein (LDL) and that soybean lipoxygenase as well as 15-lipoxygenase also oxidize low density lipoproteins (Funk et al, Trends Cardiovasc. Med. 2001; 11, 116). Another recent study reported that in the presence of deoxycholate, lipoxygenase can convert arachidonyl and linoleoyl group in phospholipids exclusively to 15 (S)-hydroperoxyeicosatetraenoic acid and 13 (S)-hydroperoxyoctadecadienoate analogs, respectively (Perez-Gilabert et al. Arch Biochem. Biophy. 1998; 354, 18). In this regard, fatty acids bound to phospholipids with ester bond may be selectively oxidized by lipoxygenase. That is, it is expected that the end part of acyl group of fatty acid derivatives bound to phospholipids can be oxidized more easily.
Lipoxygenases are ubiquitously distributed in animals and plants and have various biological functions such as inflammation mediation, signal transduction mediation etc. [G. Cirino, Biochem. Pharmacol. 55 (1998), p. 105; Farooqui et al., J. Neurochem. 69 (1997), p. 889). In mammalian systems, the direct effect of lipoxygenases on phospholipids and biomembranes (Kühn et al, J. Biol. Chem. 265 (1990) 18351-18361; Brash et al, Biochemistry 26 (1987) 5465-5471; Jung et al, Biochem. Biophys. Res. Commun. 130 (1985) 559-566; Takahashi et al, Eur. J. Biochem. 218 (1993) 165-171) suggests a role of lipoxygenases in some processes such as cellular maturation, which implies a change in the structure of the membrane (Rapoport et al, Biochim. Biophys. Acta 864 (1986) 471-495; Conrad et al, Proc. Natl. Acad. Sci. USA 89 (1992) 217-221). Additionally, soybean lipoxygenase-1 (LOX-1) is known to induce oxidation of phospholipids in low-density lipoprotein (LDL) directly with major implications for the onset of atherosclerosis, where phospholipids exist as a solubilized form (Funk et al, Trends. Cardiovasc. Med. 11 (2001) 116-124).
Lipoxygenation is highly dependent upon substrate availability. That is, prior art lipoxygenase substrates such as unsaturated fatty acids or polyunsaturated phosphatidylcholines have been used as their salt forms, e.g., sodium or ammonium form due to their fat-solubility and insolubility in a buffer solution. For measuring lipoxygenase activity, lipoxygenase substrates, linolate and arachidonic acid have to be converted to their salt forms such as Na-linolate or Na-arachidonate respectively and dispersed by a detergent added to the reaction solution. Detergents used for this purpose are Tween 20 and deoxycholate (G. Began et al. Biochemistry (1999) 38; 13920). In the presence of deoxycholate, polyunsaturated acyl moieties in phosphatidylcholine were converted by lipoxygenase to the respective hydroperoxides, although the oxidation rate was much lower for phosphatidylcholine substrate than free fatty acid substrate. For example, arachidonyl and linoleoyl moieties in phosphatidylcholine were converted to exclusively to 15 (S)-hydroperxyeicosatetraenoic acid and 13 (S)-hydroperoxyoctadecadienoate analogs, respectively, suggesting that fatty acids esterified in phospholipids can be subjected to highly specific oxygenation by lipoxygenase (Brash et al, Biochemistry 26 (1987) 5465-5471; Arai et al, Lipids 30 (1995) 135-140). Lipoxygenation of dilinoleoyl phosphatidylcholine having 2 ester bonds has to be faster than that of linoleic acid. However, it is about 4 times slower (M. Perez-Gilabert et al., Arch. Biochem. Biophys. 1998; 354, 18) since although dilinoleoyl phosphatidylcholine can be used as a LOX substrate, the reaction speed is limited for that structure being itself bi-layer.
The above-described prior art methods for measuring lipoxygenase activity have problems in that a detergent is required to be used with concentration higher than micellation concentration and that solubilization process can take a long time. In addition, when there exists LOX in tissues or cells, unsatisfied results can occur.
There is thus a need for a new method for analyzing lipoxygenase activity.
The information disclosed in this Background section is only for enhancement of understanding of the background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is already known to a person skilled in the art.